The present invention relates to a method and apparatus for determining analytes in a sample, and more particularly to a method and apparatus where the analytes are separated prior to detection.
Biomolecules may be present in several heteroforms, such as isoforms, where small changes in the molecular structure may cause great changes in the effect of the molecule. Such small structural changes may, however, be difficult to measure specifically, even with methods of high specificity, such as immunoassays, as the compounds usually will compete for the binding to a specific antibody. In our copending international (PCT) application WO 99/60402, such structural changes are discussed and a method is disclosed for measuring some of the heteroforms, for example those having the highest positive or negative charge. The method in question uses a flow matrix having an application zone for sample and, downstream thereof, a detection zone with immobilized reagent which binds analyte and where bound analyte is detected. A separation zone is provided between the sample application zone and the detection zone. In the separation zone, disturbing components or components not to be determined are bound or retarded and prevented from reaching the detection zone with the analyte. If, for example, the analyte is one of two heteroforms, the other heteroform, which is not to be determined but would compete with the analyte for binding in the binding zone, is retarded in the separation zone to permit selective detection of the analyte. There may, however, often be more than two heteroforms. For example, transferrin may exist in at least nine different isoforms, where a few of the isoforms, primarily disialo transferrin but also asialo transferrin, are important to measure for testing alcohol abuse. To be able to measure all the isoforms in a complex mixture, it has so far been necessary to separate the isoforms by column chromatography, and then analyze each fraction for the presence of an isoform by spectrophotometric or immunoassay detection depending on the concentrations of the analytes to be measured.
WO 99/30145 discloses 2-dimensional gel electrophoresis for qualitative determination of nucleic acids, proteins, carbohydrates or lipids in a sample. The gel contains a separation gel with a sample loading zone and provided in a slot within the separation gel, a detection gel having an immobilized probe for one or more target molecules. After electrophoretic separation in the separation gel in a first dimension, the gel is rotated 90 degrees and electrophoresis is performed in a second dimension to transport the target molecule to the detection zone where binding of the target molecule to the immobilized probe is detected. There is no suggestion in WO 99/30145 that heteroforms could be determined. Also, electrophoretic systems are generally rather laborious and often expensive, especially when an additional detection step is to be included in the electrophoretic system.
U.S. Pat. No. 4,469,601 discloses a method and system for multi-dimensional chromatography in a thin-layer chromatographic plate wherein a sample is separated into an array of constituents. These constituents are then separated into a second array of sub-constituents by pumping a fluid through the plate in a direction crossing the array, and the sub-constituents are detected as they flow past fixed positions in this second direction. Thin layer chromatography is, however, restricted to the separation of small (i.e. low molecular weight) molecules, and does not permit the separation of biomolecules, such as proteins, for example.
Pristoupil, T. I., Chromatog. Rev., 12 (1970) 109-125 describes the use of nitrocellulose filters in chromatography and electrophoresis. Chromatography in aqueous solution was performed with a nitrocellulose membrane in a horizontal position in a plexiglass chamber. Proteins were detected by immersing the membrane in a staining solution, and other substances were detected by usual spray or sandwich techniques. On the intact membrane, proteins having a molecular weight of the order of 105 and higher were firmly adsorbed on the membrane while peptides, amino acids and other low-molecular substances of hydrophilic character migrated with the front of the developing solution. For electrophoresis, it was necessary to impregnate the membrane with neutral detergents to prevent the high adsorption of proteins. Also immunochromatography of rabbit anti-bovine serum and immunochemically inactive normal rabbit serum on a membrane with bovine serum adsorbed thereto is described. The antigen-antibody complex gave a distinct spot at the start, while the immunochemically inactive proteins migrated without any marked adsorption. Thus, no xe2x80x9ctruexe2x80x9d chromatography of components seems to have been obtained neither in the intact (or plain) membrane nor in the antibody-coated membrane but rather either firm binding or no binding at all.
There is therefore a need for an analytical method and apparatus which permit the determination of heteroforms of biomolecules and by which assays may be performed more quickly and more easily than by the prior art methods and apparatuses, respectively.
Accordingly, it is an object of the present invention to provide a determination method which overcomes the shortcomings of the prior art methods and which readily permits quick and reliable determination of all heteroforms of biomolecules, such as isoforms, even in a complex sample. It is a further object of the present invention to provide a determination method which may be performed on a single plate, sheet or chip. Still another object of the present invention is to provide a simple and easy-to-use apparatus of plate, sheet or chip type for performing the method of the present invention.
According to the present invention, the above and other objects and advantages are obtained by a method wherein analytes (such as isoforms) in an aqueous sample are separated in a flow matrix which permits capillary force assisted fluid flow therethrough, especially a planar flow matrix such as a chromatographic membrane (e.g. an ion exchange membrane). To determine the separated analytes, this being the gist of the invention, the separated analytes are eluted from the separation area of the flow matrix in a direction substantially transverse to the separation direction to migrate to a capture zone with immobilized reactant (such as a single immobilized antibody common to all the analytes), usually in the form of a line or band, where the eluted analytes are captured. There, the analytes may be detected and determined by the addition of a detection reagent capable of binding to the captured analytes. The detection reagent may e.g. be a suitably labelled antibody directed to the isoform, such as an antibody labelled by a black-coloured particle. In the latter case, for example, the varying colour intensity along the detection line or band may be readily detected and quantified by means of a scanner.
Essentially aqueous systems are used in the separation and elution steps. xe2x80x9cEssentially aqueousxe2x80x9d means here that the system is either completely aqueous or may contain a small amount, not more than about 3%, of one or more other solvents. Preferably, about 99%, more preferably about 99.5%, usually at least about 99.9% of the essentially aqueous system is water.
Thus, in one aspect the present invention provides a method for qualititative, semi-quantitative or quantitative determination of at least two analytes in an aqueous sample containing or suspected of containing said analytes, said method comprising the steps of:
(i) providing a flow matrix comprising a separation zone extending in a first dimension thereof, and a detection zone extending in said first dimension in a spaced parallel relationship with the separation zone, said detection zone comprising an immobilized reagent capable of capturing said analytes through biospecific interaction therewith,
(ii) applying said sample to the flow matrix at or upstream of said separation zone,
(iii) initiating a first essentially aqueous fluid flow in the flow matrix along the separation zone in said first dimension to transport said analytes through said separation zone to be separated therein,
(iv) interrupting said first fluid flow and initiating a second essentially aqueous fluid flow in a second dimension of the flow matrix substantially transverse to said first dimension towards the detection zone, to transport said separated analytes to the detection zone to be captured therein by said immobilized reagent, and
(v) determining said analytes in said detection zone.
In another aspect, the present invention provides an apparatus for carrying out the method of the invention, which apparatus comprises:
(i) a flow matrix having a separation zone and a detection zone extending in a spaced parallel relationship in a first dimension of the flow matrix, wherein the detection zone comprises immobilized reagent capable of binding the analytes through specific interaction therewith,
(ii) means for initiating a first essentially aqueous fluid flow in the flow matrix along the separation zone in said first dimension of the flow matrix,
(iii) means for initiating a second essentially aqueous fluid flow in a second dimension of said flow matrix substantially transverse to the said first dimension towards the detection zone,
such that when a sample containing the analytes is introduced into to the separation zone, the analytes may be separated in the separation zone by said first fluid flow through the separation zone and transported by said second fluid flow to the detection zone where the analytes are bound to the immobilized reagent to be determined.
Preferably, the flow matrix is at least substantially planar.
The separation zone and the detection zone (which may be integral or two separate parts joined to each other) may either be arranged in the same plane of the flow matrix, or be arranged on top of each other. In the latter case, the two zones must be prevented from contacting each other such as by a removable partitioning element when the separation phase of the method of the invention is performed. Such a separating element may be a film or the like that is removed prior to performing steps (iv) and (v) above.